Endogenous metabolite

N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) and its quinone derivative, 6PPD-quinone (6PPD-Q), have been found to be prevalent in the environment, but there are currently no data on their presence in humans. Herein, we conducted the first human biomonitoring study of 6PPD and 6PPD-Q by measuring 150 urine samples collected from three different populations (general adults, children, and pregnant women) in South China. Both 6PPD and 6PPD-Q were detected in the urine samples, with detection frequencies between 60% and 100%. Urinary 6PPD-Q concentrations were significantly higher than those of 6PPD and correlated well with those of 6PPD (p < 0.01), indicating coexposure to 6PPD and 6PPD-Q in humans. In vitro metabolic experiments demonstrated rapid depletion of 6PPD by human liver microsomes, which should be responsible for the lower concentrations of 6PPD in human urine. Additionally, pregnant women exhibited apparently higher concentrations of 6PPD and 6PPD-Q (median 0.068 and 2.91 ng/mL, respectively) than did adults (0.018 and 0.40 ng/mL) and children (0.015 and 0.076 ng/mL). The high daily urinary excretion of 6PPD-Q in pregnant women was estimated to be 273 (ng/kg bw)/day. Considering that 6PPD-Q was a lethal toxicant to multiple aquatic species, the potential human health risks posed by its long-term exposure require urgent attention[1].

Daily Excretion of 6PPD and 6PPD-Q in Urine [1]
Based on the urinary analyte concentrations measured in the three populations, the daily excretion of 6PPD and 6PPD-Q in urine was estimated using a model as described elsewhere, (49−51) i.e., daily excretion of 6PPD and 6PPD-Q in urine ((ng/kg bw)/day) = urinary analyte concentration (ng/mL) × daily excretion volume of urine (mL/day)/body weight (kg). In our estimation, values for daily excretion volume of urine were adopted from refs (50and51): 1700 mL/day for adults, 660 mL/day for children, and 2000 mL/day for pregnant women. Values for body weight were based on the average body weight obtained in our questionnaires: 60 kg for adults, 23 kg for children, and 64 kg for pregnant women (Table S2). Median and 95th concentrations of 6PPD and 6PPD-Q were used to calculate the median and high daily excretion, respectively. As shown in Figure 2, as expected, the daily excretion of 6PPD-Q in urine of adults, children, and pregnant women (median 11.3, 2.18, 90.9 (ng/kg bw)/day, respectively) was evidently higher than its parent 6PPD (median 0.51, 0.43, and 2.13 (ng/kg bw)/day, respectively). In addition, pregnant women had higher daily excretion of 6PPD and 6PPD-Q than did adults and children, and the high daily excretion of 6PPD-Q in urine of pregnant women was up to 247 (ng/kg bw)/day.
It should be pointed out that due to the difficulties in collecting 24-h urine samples, the estimates of daily excretion were limited by the reliance on spot morning urine samples, as there may be daily variance in urinary 6PPD and 6PPD-Q levels. Therefore, although many previous studies suggest that early morning urine is a feasible alternative to 24-h collections for estimating personal exposure, these results should be considered as a preliminary estimation. Despite the limitation, the daily urinary excretion of 6PPD and 6PPD-Q in these populations should be of concern because it largely reflects their internal exposure dose. Given that a portion of 6PPD and 6PPD-Q may also be excreted through feces and exhaled air like some other pollutants, the actual exposure dose of 6PPD and 6PPD-Q may even be higher than our estimates. Although the dose is unlikely to exceed a proposed reference dose (RfD) of 26000 (ng/kg bw)/day calculated for 6PPD, (56) the potential health risk posed by long-term exposure to 6PPD-Q requires considerable attention, as 6PPD-Q has been proven to be more toxic to aquatic organisms than 6PPD from the individual species to the tissue levels. In future studies, additional biomonitoring investigations on 6PPD and 6PPD-Q are needed to better elucidate their internal exposure in humans. More toxicological and epidemiological studies of 6PPD and 6PPD-Q are also recommended to shed light on their potential health risks.

[1]. First report on the occurrence of N-(1, 3-dimethylbutyl)-N’-phenyl-p-phenylenediamine (6PPD) and 6PPD-quinone as pervasive pollutants in human urine from south China. Environ Sci Technol Lett, 2022, 9(12): 1056-1062.

p-Phenylenediamine (PPD) compounds are an important class of synthetic antioxidants, once widely used as additives in various rubber products (such as tires, footwear, and even food contact materials). Among PPD compounds, N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) is one of the most commonly used compounds and has been listed as a high-yield (HPV) chemical. In 2001, global 6PPD production was estimated at 130,000 tons. By 2020, China alone had an annual production of 200,000 tons, accounting for nearly 54% of the total production of rubber antioxidants. However, such high production has raised concerns. A recent study by Tian et al. suggests that 6PPD may pose safety hazards. They found that 6-p-phenylenediamine (6PPD) can be oxidized in the environment to produce a toxic quinone derivative called 6PPD-quinone (6PPD-Q), which is the culprit behind the acute death of silver salmon (Oncorhynchus kisutch) in the Pacific Northwest. (6) Due to the widespread use of 6PPD, this discovery has sparked widespread concern in the scientific community about the environmental pollution and toxic effects of 6PPD and its quinone derivative 6PPD-Q. [1] In recent years, 6PPD and 6PPD-Q have been found in a variety of environmental media, including atmospheric particulate matter, indoor dust (5,11,12), road dust, playground dust, roadside soil, runoff and surface water. (16-18) In most cases, 6PPD and 6PPD-Q coexist in these environmental media at similar concentrations. For example, Zhang et al. found that when the concentrations of 6PPD and 6PPD-Q were 100% higher than the concentration of 6PPD, ... Studies have shown that dioctyl 6-terephthalate (6PPD) and dioctyl 6-terephthalate-Q (6PPD-Q) are widely distributed in fine particulate matter (PM2.5) in six Chinese cities, and the atmospheric concentrations of 6PPD (median 0.9–8.4 pg/m3) are similar to those of 6PPD-Q (median 1.7–6.7 pg/m3). Hiki et al. reported the coexistence of 6PPD and 6PPD-Q in road dust in Tokyo, Japan, and also observed comparable concentrations of 6PPD (45–1175 ng/g) and 6PPD-Q (116–1238 ng/g) in dust samples. [1] In addition to their widespread presence in the environment, emerging evidence suggests that 6PPD and 6PPD-Q are toxic. (6,19-24) For example, 6-p-phenylenediamine (6PPD) has been shown to be toxic to Pimephales promelas and freshwater mussels (Lampsilis siliquoidea). In addition to its high toxicity to coho salmon as reported by Tian et al. (6,21), 6PPD-Q has also been shown to be toxic to zebrafish larvae, with a 24-hour LC50 of 308.67 μg/L. Recent studies have found that 6PPD and 6PPD-Q can cause anxiety-like behavior and balance disorders in zebrafish. Although the aquatic toxicity of 6PPD and 6PPD-Q has become clearer, their adverse effects on mammals and humans remain unclear. In particular, internal human exposure to 6PPD and 6PPD-Q may pose a potential health risk. However, although the prevalence of dioctyl terephthalate (6PPD) and dioctyl terephthalate-Q (6PPD-Q) in the environment has been confirmed, their presence, concentration and exposure in humans remain unclear. [1] To fill this knowledge gap, we conducted the first human biomonitoring study on 6PPD and 6PPD-Q by detecting urine samples. Considering that urine plays an important role in the excretion of environmental pollutants and that urine samples are relatively easy to collect, store and process, we chose urine samples for our study. We collected a total of 150 urine samples from three different populations in southern China (general adults, children and pregnant women) and analyzed the levels of 6PPD and 6PPD-Q in them. The main objectives of this study were: (1) to determine the presence of 6PPD and 6PPD-Q in human urine; (2) to compare the levels of 6PPD and 6PPD-Q in different populations in southern China; and (3) to provide baseline information on human exposure to 6PPD and 6PPD-Q. The results of this study will greatly enhance our understanding of human exposure to 6PPD and 6PPD-Q. [1]

C18H22N2O2

298.38

298.168

2754428-18-5

154926030

Pink to red solid powder

4.1

2

4

6

22

485

0

C1(NC(CC(C)C)C)C(C=C(C(C=1)=O)NC1C=CC=CC=1)=O

UBMGKRIXKUIXFQ-UHFFFAOYSA-N

InChI=1S/C18H22N2O2/c1-12(2)9-13(3)19-15-10-18(22)16(11-17(15)21)20-14-7-5-4-6-8-14/h4-8,10-13,19-20H,9H2,1-3H3

2-anilino-5-(4-methylpentan-2-ylamino)cyclohexa-2,5-diene-1,4-dione

6PPD-quinone; 2754428-18-5; 6PPD-Q; 2-((4-Methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione; 2,5-Cyclohexadiene-1,4-dione, 2-[(1,3-dimethylbutyl)amino]-5-(phenylamino)-; 6PPD quinone; 6PPD-Quinone; 2-((4-Methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione; 6PPD-quinone; 6PPD-Quinone; 2-[(1,3-Dimethylbutyl)amino]-5-(phenylamino)-2,5-cyclohexadiene-1,4-dione (ACI); 2,5-Cyclohexadiene-1,4-dione, 2-[(1,3-dimethylbutyl)amino]-5-(phenylamino)- (ACI); G8MFB8G7B6;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)